蛋白质组学原理及应用案例 - CAS

1 2019- 2000-2004 2004-2006 2006-2010 2011-2013 2013-2018 WB MRMHR/PRM VS LC-MS/MS Bottom-up proteomics or Shotgun proteomics MS2 @ .. LFQ TMT/iTRAQ SWATH .. @ MRMHR/PRM @ WB (Discovery proteomics ) --- LFQ/SWATH--- SILAC/ / 11.

2 B1N QWFFSK y1b2NQ WFFSK y2b3 NQW FFSK y3b4 NQWF FSK y4b5 NQWFF SK y5b6 NQWFFS K y6 Proteindatabase : ng/mL : / SILAC/ /TMT/iTRAQ SWATH VS / / IVS IISilence/inhibition/over-expression VS Candidate proteins (qPCR/WB/ELISA/PRM) (silence/mutation/inhibition) GO PPI/ Top changed/prediction /references Label-free quantification 14 Label-freequantification example: Murine Liver HC/KC/LSEC/CHC/HSC MaxLFQ Perseus, R Cell Metabolism: 20, 1076 1087, 2014 11520 + / + + + PRM AE-MS (Affinity-EnrichmentMS) Molecular & Cellular Proteomics 14, 120 135, 2015AE-MS AP-MS 6iTRAQ(isobaric tags for relative and absolute quantification )iTRAQ reagentiTRAQ labeled peptide8plex: 113/114/115/116/117/118/119/121iTRAQ workflowiTRAQ example:dietary protein limitation response PL iTRAQ 5275 202 Scientific Reports.

3 6, 36888, 2016 SWATH/DIA400-1200 m/z =800 m/z = 25 m/z 32 windows Sequential Window Acquisition of all Theoretical mass spectra 2012 RuediAebersold AB TripleTOF5600 SWATH/DIA Targeted extraction of quantitative information from the acquired DIA data with the use of libraries containing retention time and fragmentation information for the desired peptide species. [Skyline/Spectronaut] DIA data pseudo tandem MS spectra (untargeted) database search Quantification by targeted extraction[DIA-Umpire/Group-DIA/MSPLIT-D IA] LPS (Targeted proteomics) discovery-based investigation / hypothesis-driven investigation MS strategy: Targeted VS.

4 Large-scale MRM & PRMMRM/SRMMRMHRPRM Authorswhosubmitpaperscontainingquantita tiveproteindatageneratedviaMSarefrequent lyaskedbyreviewerstovalidatesomeoftheval ueswithWesternblotting. Unfortunately,thequalityofquantitativeda taobtainedviaWesternblottingisnotcompara bletothatobtainedwithSRM. Wepositthattherequesttovalidatequantitat iveMSdatabyWesternblottingisnolongerjust ified. Infact,itmaybetimeto turnthetables 4 1 mol = 103mmol = 106 mol = 109 nmol = 1012 pmol = 1015 fmol = 1018 amolPRM Western Blot / / WB PRM / DDA (Shotgun, IDA, topN) SWATH/DIA Targeted method (MRM, SRM.)

5 PRM, MRMHR) TMT SILAC DIA PRM PD Label-free MaxQuant Thermo Q ExactiveHF-X Thermo LTQ Orbitrap Elite Orbitrap 14 SCI 30 5 2 8

6 SDS-PAGE IP TMT iTRAQ Dimethylation SILAC IMAC/TiO2